1. Field of The Invention
This invention relates to a method for culturing Bordetella pertussis. The present invention also relates to a pertussis toxoid. Further, the present invention is concerned with a vaccine comprising the pertussis toxoid as an active ingredient. The method of the present invention is advantageous for producing a culture which contains an increased amount of a mixed antigen comprising pertussis toxin and pertussis filamentous hemagglutinin. From the culture, the mixed antigen can easily be isolated. The pertussis toxoid of the present invention is readily obtained from the mixed antigen and, therefore, can be produced easily at low cost. The pertussis toxoid of the present invention is extremely effective as an active ingredient for a pertussis vaccine and a diagnostic agent for pertussis.
2. Discussion of Related Art
Pertussis is an acute respiratory infectious disease caused by the infection of Bordetella pertussis. The Pertussis affects the bronchus and bronchiole and is characterized by explosive coughing ending in a whooping inspiration. While coughing is characteristic of the disease and is vulgarly said to last for a hundred days, the explosive coughing, in fact, lasts for several weeks. Particularly, in the case of an infant suffering from pertussis, an apnea-paroxysmal cough occurs and is sometimes accompanied with a spasm. For preventing this disease, in Japan, since 1949, the inoculation of pertussis vaccine to infants in the early period after birth has been carried out. Conventional pertussis vaccines contain inactivated cells of the bacterium as an active ingredient and, therefore, their inoculation tends to cause unfavorable side effects. Well known side effects include, for example, various local reactions at the site of the inoculation of a pertussis vaccine, systemic symptoms such as fever, moroseness, vomiting and diarrhea, temporary nervous spasm and neurogenic shock, and severe encephalitis and the like. Since, morbidity and mortality from pertussis have decreased noticeably in recent years, due to improvements in living conditions and the development of chemotherapies, the above-mentioned side effects caused by inoculation of the vaccine have become an even more serious problem than the danger of pertussis infection. However, it is very dangerous to stop vaccinating for pertussis in order to avoid those side effects. This is apparent from the past bitter experience that many infant patients died of pertussis in Japan and England owing to natural prevalence of pertussis which was caused by the temporary stop of vaccination. Accordingly, the inoculation of pertussis vaccine is still considered to be important and necessary.
Heretofore, there have been reported many substances as a protective antigen, biologically active substance or component of Bordetella pertussis. As such substances, there may be mentioned, for example, a heat-labile toxin or a dermonecrotic toxin; a heat-labile agglutinogen; an endotoxin or a lipopolysaccharide; a filamentous hemagglutinin (F-HA); a histamine sensitizing factor (HSF); a leukocytosis or lymphocytosis promoting factor (LPF); a pertussis toxin (PT) which is also called lymphocytosis promoting factor hemagglutinin (LPF-HA); an adjuvant factor; an adenylate cyclase; an islet-activating factor; an outermembrane protein and the like. Of those substances, up to now, the filamentous hemagglutinin (hereinafter often referred to as "F-HA") and pertussis toxin (hereinafter often referred to as "PT") have been put to practical use as the active ingredient of a pertussis toxoid or a pertussis component vaccine.
As mentioned before, it is known that conventional vaccines have various side effects because the vaccines contain inactivated cells of the bacterium as an active ingredient. Accordingly, there has been a world-wide desire to develop a safe and potent vaccine having a uniform quality without the above-mentioned side effects (Journal of American Medical Association, 125(2), 51-252, 1984; Bulletin of the World Health Organization, 63(2), 241-248, 1985). In Japan, a pertussis toxoid (a purified adsorbed pertussis vaccine) which contains as an active ingredient a substance obtained by detoxifying a protective antigen fraction (F-HA and PT) derived from Bordetella pertussis has already been approved by the Ministry of Health and Welfare, Japan, and has been marketed since Autumn 1981. With respect to the techniques for preparation of the vaccine, there are disclosed in, for example, Japanese Patent Publication No. 57-5203, U.S. Pat. No. 4,455,297, U.S. Pat. No. 4,500,639, Japanese Patent Application Laid-Open Specification No. 58-222032, Japanese Patent Application Laid-Open Specification No. 59-175439, European Patent Application Laid-Open Specification No. 121249, European Patent Application Laid-Open Specification No. 140386, Japanese Patent Application Laid-Open Specification No. 60-218326, Japanese Patent Application Laid-Open Specification No. 60-226822, Japanese Patent Publication No. 60-28277, Japanese Patent Application Laid-Open Specification No. 60-237023, Japanese Patent Application Laid-Open Specification No. 61-53224, European Patent Application Laid-Open Specification No. 175841, Japanese Patent Application Laid-Open Specification No. 62-5922, U.S. Pat. No. 4,029,766, U.S. Pat. No. 4,551,429, etc. Further, with respect to the preparation of a component vaccine by the use of a substance derived from Bordetella pertussis as an active ingredient, there are known, for example, the use of cell wall (Japanese Patent Publication No. 56-47167), the use of islet-activating factor (Japanese Patent Application Laid-Open Specification No. 59-110626), the use of outermembrane protein (European Patent Laid-Open Specification No. 4137, European Patent Laid-Open Specification No. 80021), the use of a fraction having adenylate cyclase activity (European Patent Application Laid-Open Specification No. 162639), the use of an endotoxin (French Patent Application Laid-Open Specification No. 2356429), etc. However, the above-mentioned customary techniques each have at least one of the following disadvantages.
(1) The production cost is high because expensive materials are needed. PA1 (2) The yield of an active ingredient for a vaccine is low. PA1 (3) The safety, potency, uniformity in quality and stability of a vaccine produced is not satisfactory. PA1 (1) Cyclodextrin to be used is expensive. PA1 (2) The produced PT and F-HA are in a state which includes the cyclodextrin contained in the medium and it is difficult to separate them from the cyclodextrin and highly purify them. That is, such a technique is not economic because expensive materials and expensive purification apparatuses are needed and the procedures involved therein are complicated and troublesome. Therefore, it is difficult to obtain a pertussis vaccine having safety and uniformity in quality. Further, in the above-mentioned U.S. Pat. No. 4,551,429, it is disclosed that a polyvinyl alcohol is added to a basal medium of Bordetella pertussis for the same purpose as described above. However, in this reference, there is no description of a method for purifying PT and F-HA to high degree and of the quality of the PT and F-HA produced and, therefore, it is questionable that the vaccine obtained according to this reference contains high purity PT and F-HA and is uniform in quality.
For Example, in U.S. Pat. No. 4,500,639, Japanese Patent Application Laid-Open Specification No. 59-175439, European Patent Application Laid-Open Specification No. 121249 and Japanese Patent Publication No. 60-28277 mentioned above, it is disclosed that in order to increase the yields of PT and F-HA, cyclodextrin which is known as a host compound capable of forming inclusion complexes is added to a basal medium for culturing Bordetella pertussis. However, this technique has the following disadvantages.
As is generally known, in 1979, WHO (World Health Organization) started an Expanded Program on Immunization (EPI) and is now carrying out this program. The object of the program is to give 6 kinds of vaccines in total, namely, pertussis vaccine, tetanus vaccine, diphtheria vaccine, tuberculosis vaccine, polio vaccine and measles vaccine to all the infants in the world until 1990 (WHO Chronicle, 36(4) 131-152, 1982). In order to fulfill this program, the WHO urges the development of vaccines which are excellent in stability and heat resistance and can be used in the tropics in which refrigerators are not wide spread, and put them to practical use [WHO Technical Report Series, No. 673, 15, (1982)]. Furthermore, vaccines, the titers of which do not decrease even when they are frozen, are also extremely useful because the vaccines are also used in cold districts. Therefore, a world wide desire exists to develop highly stable vaccines which are excellent in both heat resistance and cold resistance and, therefore, can be used at an atmospheric temperature in any of the tropics and the cold districts. With respect to heat-resistant vaccines, dried vaccines such as a live virus vaccine, an inactivated virus vaccine, a mixed acellular vaccine comprising an inactivated bacterium and a toxoid (Japanese Patent Application Laid-Open Specification No. 59-19682) and the like are known. However, the above-mentioned dried vaccines are poor in stability such that after storage at 37.degree. C. for about a month, titers decrease as much as 10 to 50% relative to the titers of the vaccines before the storage. Hence, it has been strongly desired to develop a vaccine which is extremely excellent in heat resistance and cold resistance and stable such that the titer of the vaccine is scarcely decreased even when preserved, for example, at a temperature of from about -20.degree. C. to about 50.degree. C. for several months.
Furthermore, a pertussis toxoid obtained by detoxifying a pertussis toxin using formalin is now available on the market, but such a toxoid has a serious disadvantage that the toxoid is apt to revert to a toxic state during storage and, therefore, after storage the safeness of the toxoid can no longer be assured. In order to prevent the above-mentioned reversion of the toxoid to a toxic state, it has been proposed that instead of formalin, carbodiimide is used as a detoxifying agent (Japanese Patent Application Laid-open Specification No. 62-5922). However, the use of carbodiimide is questionable because the safeness of carbodiimide has not been confirmed.
It is earnestly desired in the art to eliminate the above-mentioned drawbacks and disadvantages of the conventional vaccines and develop an improved pertussis vaccine.